RELATED APPLICATIONS
This application claims benefit of US Provisional Patent Application Number 60/052,758, filed
July 1, 1997.
FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides and polypeptides, and their
production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, the
invention relates to polynucleotides and polypeptides of the gidA family, as well as their variants,
hereinafter referred to as "gidA1," "gidA1 polynucleotide(s)," and "gidA1 polypeptide(s)" as the case
may be.
BACKGROUND OF THE INVENTION
It is particularly preferred to employ Staphylococcal genes and gene products as targets for the
development of antibiotics. The Staphylococci make up a medically important genera of microbes.
They are known to produce two types of disease, invasive and toxigenic. Invasive infections are
characterized generally by abscess formation affecting both skin surfaces and deep tissues. S. aureus is
the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic
thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three
clinical conditions resulting from the toxigenic properties of Staphylococci. The manifestation of
these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia. These
conditions include: Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome.
The frequency of Staphylococcus aureus infections has risen dramatically in the past few
decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an
increasing population of people with weakened immune systems. It is no longer uncommon to isolate
Staphylococcus aureus strains which are resistant to some or all of the standard antibiotics. This
phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines,
drug screening methods, and diagnostic tests for this organism.
Moreover, the drug discovery process is currently undergoing a fundamental revolution as it
embraces "functional genomics," that is, high throughput genome- or gene-based biology. This
approach is rapidly superseding earlier approaches based on "positional cloning" and other methods.
Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of
potential interest from the many molecular biology databases now available as well as from other
sources. There is a continuing and significant need to identify and characterize further genes and other
polynucleotides sequences and their related polypeptides, as targets for drug discovery.
Clearly, there exists a need for polynucleotides and polypeptides, such as the gidA1
embodiments of the invention, that have a present benefit of, among other things, being useful to screen
compounds for antimicrobial activity. Such factors are also useful to determine their role in
pathogenesis of infection, dysfunction and disease. There is also a need for identification and
characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or
correct such infection, dysfunction and disease.
Certain of the polypeptides of the invention possess significant amino acid sequence homology
to a known gidA protein.
SUMMARY OF THE INVENTION
The present invention relates to gidA1, in particular gidA1 polypeptides and gidA1
polynucleotides, recombinant materials and methods for their production. In another aspect, the
invention relates to methods for using such polypeptides and polynucleotides, including treatment of
microbial diseases, amongst others. In a further aspect, the invention relates to methods for
identifying agonists and antagonists using the materials provided by the invention, and for treating
microbial infections and conditions associated with such infections with the identified agonist or
antagonist compounds. In a still further aspect, the invention relates to diagnostic assays for detecting
diseases associated with microbial infections and conditions associated with such infections, such as
assays for detecting gidA1 expression or activity.
Various changes and modifications within the spirit and scope of the disclosed invention will
become readily apparent to those skilled in the art from reading the following descriptions and from
reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The invention relates to gidA1polypeptides and polynucleotides as described in greater detail
below. In particular, the invention relates to polypeptides and polynucleotides of a gidA1 of
Staphylococcus aureus, which is related by amino acid sequence homology to gidA polypeptide. The
invention relates especially to gidA1 having the nucleotide and amino acid sequences set out in Table 1
as SEQ ID NO: 1 or 3 and SEQ ID NO: 2 or 4 respectively.
Deposited materials
A deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the
National Collections of Industrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive,
Aberdeen AB2 1RY, Scotland on 11 September 1995 and assigned NCIMB Deposit No. 40771, and
referred to as Staphylococcus aureus WCUH29 on deposit. . The Staphylococcus aureus strain deposit
is referred to herein as "the deposited strain" or as "the DNA of the deposited strain."
The deposited strain contains the full length gidA1 gene. The sequence of the polynucleotides
contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded
thereby, are controlling in the event of any conflict with any description of sequences herein.
The deposit of the deposited strain has been made under the terms of the Budapest Treaty on
the Intemational Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The
strain will be irrevocably and without restriction or condition released to the public upon the issuance
of a patent. The deposited strain is provided merely as convenience to those of skill in the art and is not
an admission that a deposit is required for enablement, such as that required under 35 U.S.C. §112.
A license may be required to make, use or sell the deposited strain, and compounds derived
therefrom, and no such license is hereby granted.
In one aspect of the invention there is provided an isolated nucleic acid molecule encoding a
mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is
contained in the deposited strain. Further provided by the invention are gidA1polynucleotide
sequences in the deposited strain, such as DNA and RNA, and amino acid sequences encoded thereby.
Also provided by the invention are gidA1 polypeptide and polynucleotide sequences isolated from the
deposited strain.
Polypeptides
gidA1 polypeptide of the invention is substantially phylogenetically related to other proteins of
the gidA family.
In one aspect of the invention there are provided polypeptides of Staphylococcus aureus
referred to herein as "gidA1" and "gidA1 polypeptides" as well as biologically, diagnostically,
prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the
same.
Among the particularly preferred embodiments of the invention are variants of gidA1
polypeptide encoded by naturally occurring alleles of the gidA1 gene.
The present invention further provides for an isolated polypeptide which:
(a) comprises or consists of an amino acid sequence which has at least 70% identity, preferably at
least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity,
most preferably at least 97-99% or exact identity, to that of SEQ ID NO:2 over the entire length of
SEQ ID NO:2; (b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a
polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more
preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at
least 97-99% or exact identity to SEQ ID NO:1 over the entire length of SEQ ID NO:1; (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide
sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity,
more preferably at least 90% identity, yet more preferably at least 95% identity, even more
preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO:2, over the
entire length of SEQ ID NO:2; (d) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a
polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more
preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at
least 97-99% or exact identity, to SEQ ID NO:1 over the entire length of SEQ ID NO:3; (e) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a
polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more
preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at
least 97-99% or exact identity to SEQ ID NO:3 over the entire length of SEQ ID NO:3; or (f) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a
polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least
80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even
more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO:4, over
the entire length of SEQ ID NO:4; (g) comprises or consists of an amino acid sequence which has at least 70% identity, preferably at
least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity,
most preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO:2 over
the entire length of SEQ ID NO:4.
The polypeptides of the invention include a polypeptide of Table 1 [SEQ ID NO:2 or 4] (in
particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have
the biological activity of gidA1, and also those which have at least 70% identity to a polypeptide of
Table 1 [SEQ ID NO:1 or 3]or the relevant portion, preferably at least 80% identity to a polypeptide of
Table 1 [SEQ ID NO:2 or 4and more preferably at least 90% identity to a polypeptide of Table 1 [SEQ
ID NO:2 or 4] and still more preferably at least 95% identity to a polypeptide of Table I [SEQ ID NO:2
or 4] and also include portions of such polypeptides with such portion of the polypeptide generally
containing at least 30 amino acids and more preferably at least 50 amino acids.
The invention also includes a polypeptide consisting of or comprising a polypeptide of the
formula:
X-(R1)m-(R2)-(R3)n-Y
wherein, at the amino terminus, X is hydrogen, a metal or any other moiety described herein for
modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety
described herein for modified polypeptides, R1 and R3 are any amino acid residue or modified amino
acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero,
and R2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from
Table 1 or modified forms thereof. In the formula above, R2 is oriented so that its amino terminal
amino acid residue is at the left, covalently bound to R1, and its carboxy terminal amino acid residue is
at the right, covalently bound to R3. Any stretch of amino acid residues denoted by either R1 or R3,
where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a
heteropolymer. Other preferred embodiments of the invention are provided where m is an integer
between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
It is most preferred that a polypeptide of the invention is derived from Staphylococcus aureus,
however, it may preferably be obtained from other organisms of the same taxonomic genus. A
polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic
family or order.
A fragment is a variant polypeptide having an amino acid sequence that is entirely the same as
part but not all of any amino acid sequence of any polypeptide of the invention. As with gidA1
polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they
form a part or region, most preferably as a single continuous region in a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a portion of an amino
acid sequence of Table 1 [SEQ ID NO:2 or 4], or of variants thereof, such as a continuous series of
residues that includes an amino- and/or carboxyl-terminal amino acid sequence. Degradation forms of
the polypeptides of the invention produced by or in a host cell, particularly a Staphylococcus aureus,
are also preferred. Further preferred are fragments characterized by structural or functional attributes
such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-fonning
regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions,
hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming
regions, substrate binding region, and high antigenic index regions.
Further preferred fragments include an isolated polypeptide comprising an amino acid
sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid
sequence of SEQ ID NO: 2, or an isolated polypeptide comprising an amino acid sequence having
at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid
sequence of SEQ ID NO:2.
Also preferred are biologically active fragments which are those fragments that mediate
activities of gidA1, including those with a similar activity or an improved activity, or with a decreased
undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal,
especially in a human. Particularly preferred are fragments comprising receptors or domains of
enzymes that confer a function essential for viability of Staphylococcus aureus or the ability to initiate,
or maintain cause Disease in an individual, particularly a human.
Fragments of the polypeptides of the invention may be employed for producing the
corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed
as intermediates for producing the full-length polypeptides of the invention.
In addition to the standard single and triple letter representations for amino acids, the term
"X" or "Xaa" may also be used in describing certain polypeptides of the invention. "X" and "Xaa"
mean that any of the twenty naturally occurring amino acids may appear at such a designated
position in the polypeptide sequence.
Polynucleotides
It is an object of the invention to provide polynucleotides that encode gidA1 polypeptides,
particularly polynucleotides that encode the polypeptide herein designated gidA1.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region
encoding gidA1 polypeptides comprising a sequence set out in Table 1 [SEQ ID NO:1 or 3] which
includes a full length gene, or a variant thereof. The Applicants believe that this full length gene is
essential to the growth and/or survival of an organism which possesses it, such as Staphylococcus
aureus.
As a further aspect of the invention there are provided isolated nucleic acid molecules
encoding and/or expressing gidA1 polypeptides and polynucleotides, particularly Staphylococcus
aureus gidA1 polypeptides and polynucleotides, including, for example, unprocessed RNAs,
ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments of the
invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful
polynucleotides and polypeptides, and variants thereof, and compositions comprising the same.
Another aspect of the invention relates to isolated polynucleotides, including at least one full
length gene, that encodes a gidA1 polypeptide having a deduced amino acid sequence of Table 1 [SEQ
ID NO:2 or 4] and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention there is a gidA1 polypeptide
from Staphylococcus aureus comprising or consisting of an amino acid sequence of Table 1 [SEQ ID
NO:2 or 4], or a variant thereof.
Using the information provided herein, such as a polynucleotide sequence set out in Table 1
[SEQ ID NO:1 or 3], a polynucleotide of the invention encoding gidA1 polypeptide may be obtained
using standard cloning and screening methods, such as those for cloning and sequencing chromosomal
DNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells as starting material,
followed by obtaining a full length clone. For example, to obtain a polynucleotide sequence of the
invention, such as a polynucleotide sequence given in Table 1 [SEQ ID NO: 1 or 3], typically a
library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E.coli or some other
suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived
from a partial sequence. Clones carrying DNA identical to that of the probe can then be
distinguished using stringent hybridization conditions. By sequencing the individual clones thus
identified by hybridization with sequencing primers designed from the original polypeptide or
polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions
to determine a full length gene sequence. Conveniently, such sequencing is performed, for example,
using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are
described by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A
LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded
DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to obtain
a full length gene sequence. Illustrative of the invention, each polynucleotide set out in Table 1 [SEQ
ID NO:1 or 3] was discovered in a DNA library derived from Staphylococcus aureus WCUH 29.
Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1 or 3] contains an open
reading frame encoding a protein having about the number of amino acid residues set forth in Table 1
[SEQ ID NO:2 or 4] with a deduced molecular weight that can be calculated using amino acid residue
molecular weight values well known to those skilled in the art. The polynucleotide of SEQ ID NO: 1,
between nucleotide number 109 and the stop codon which begins at nucleotide number 1984 of SEQ ID
NO:1, encodes the polypeptide of SEQ ID NO:2.
In a further aspect, the present invention provides for an isolated polynucleotide comprising or
consisting of:
(a) a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more
preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at
least 97-99% or exact identity to SEQ ID NO:1 over the entire length of SEQ ID NO:1; (b) a polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at
least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity,
even more preferably at least 97-99% or 100% exact, to the amino acid sequence of SEQ ID NO:2,
over the entire length of SEQ ID NO:2; (c) a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more
preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at
least 97-99% or 100% identity, to SEQ ID NO:1 over the entire length of SEQ ID NO:3; (d) a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more
preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at
least 97-99% or exact identity to SEQ ID NO:3 over the entire length of SEQ ID NO:3; or (e) a polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at
least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity,
even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO:4,
over the entire length of SEQ ID NO:4.
A polynucleotide encoding a polypeptide of the present invention, including homologs and
orthologs from species other than Staphylococcus aureus, may be obtained by a process which
comprises the steps of screening an appropriate library under stringent hybridization conditions with a
labeled or detectable probe consisting of or comprising the sequence of SEQ ID NO: 1 or 3 or a
fragment thereof; and isolating a full-length gene and/or genomic clones containing said polynucleotide
sequence.
The invention provides a polynucleotide sequence identical over its entire length to a coding
sequence (open reading frame) in Table 1 [SEQ ID NO:1 or 3]. Also provided by the invention is a
coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence
for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a
sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence. The
polynucleotide of the invention may also contain at least one non-coding sequence, including for
example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated
sequences, termination signals (such as rho-dependent and rho-independent termination
signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and
polyadenylation signals. The polynucleotide sequence may also comprise additional coding sequence
encoding additional amino acids. For example, a marker sequence that facilitates purification of the
fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a
hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.
Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984),
both of which may be useful in purifying polypeptide sequence fused to them. Polynucleotides of the
invention also include, but are not limited to, polynucleotides comprising a structural gene and its
naturally associated sequences that control gene expression.
A preferred embodiment of the invention is a polynucleotide consisting of or comprising
nucleotide 109 to the nucleotide immediately upstream of, or including, nucleotide 1984 set forth in
SEQ ID NO:1 of Table 1, both of which encode the gidA1 polypeptide.
The invention also includes a polynucleotide consisting of or comprising a polynucleotide of
the formula:
X-(R1)m-(R2)-(R3)n-Y
wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or
together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or
a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of R1
and R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer
between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R2 is a nucleic acid
sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence
selected from Table 1 or a modified nucleic acid sequence thereof. In the polynucleotide formula
above, R2 is oriented so that its 5' end nucleic acid residue is at the left, bound to R1, and its 3' end
nucleic acid residue is at the right, bound to R3. Any stretch of nucleic acid residues denoted by
either R1 and/or R2, where m and/or n is greater than 1, may be either a heteropolymer or a
homopolymer, preferably a heteropolymer. Where, in a preferred embodiment, X and Y together
define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide,
which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the
second strand is complementary. In another preferred embodiment m and/or n is an integer between
1 and 1000. Other preferred embodiments of the invention are provided where m is an integer between
1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
It is most preferred that a polynucleotide of the invention is derived from Staphylococcus
aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus. A
polynucleotide of the invention may also be obtained, for example, from organisms of the same
taxonomic family or order.
The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides
that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and
more particularly a polypeptide of the Staphylococcus aureus gidA1 having an amino acid sequence set
out in Table 1 [SEQ ID NO:2 or 4]. The term also encompasses polynucleotides that include a single
continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides
interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an
integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with
additional regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described herein that encode
variants of a polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4].
Fragments of a polynucleotides of the invention may be used, for example, to synthesize full-length
polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding gidA1 variants, that
have the amino acid sequence of gidA1 polypeptide of Table 1 [SEQ ID NO:2 or 4] in which several, a
few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or
added, in any combination. Especially preferred among these are silent substitutions, additions and
deletions, that do not alter the properties and activities of gidA1 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at least 70%
identical over their entire length to a polynucleotide encoding gidA1 polypeptide having an amino acid
sequence set out in Table 1 [SEQ ID NO:2 or 4], and polynucleotides that are complementary to such
polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is
at least 80% identical over its entire length to a polynucleotide encoding gidA1 polypeptide and
polynucleotides complementary thereto. In this regard, polynucleotides at least 90% identical over
their entire length to the same are particularly preferred, and among these particularly preferred
polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97%
are highly preferred among those with at least 95%, and among these those with at least 98% and at
least 99% are particularly highly preferred, with at least 99% being the more preferred.
Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the
same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ID
NO:1 or 3].
In accordance with certain preferred embodiments of this invention there are provided
polynucleotides that hybridize, particularly under stringent conditions, to gidA1 polynucleotide
sequences, such as those polynucleotides in Table 1.
The invention further relates to polynucleotides that hybridize to the polynucleotide sequences
provided herein. In this regard, the invention especially relates to polynucleotides that hybridize under
stringent conditions to the polynucleotides described herein. As herein used, the terms "stringent
conditions" and "stringent hybridization conditions" mean hybridization occurring only if there is at
least 95% and preferably at least 97% identity between the sequences. A specific example of stringent
hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide,
5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's
solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA,
followed by washing the hybridization support in 0.1x SSC at about 65°C. Hybridization and wash
conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein.
Solution hybridization may also be used with the polynucleotide sequences provided by the
invention.
The invention also provides a polynucleotide consisting of or comprising a polynucleotide
sequence obtained by screening an appropriate library containing the complete gene for a
polynucleotide sequence set forth in SEQ ID NO: 1 or 3 under stringent hybridization conditions with
a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO: 1 or 3 or a
fragment thereof; and isolating said polynucleotide sequence. Fragments useful for obtaining such a
polynucleotide include, for example, probes and primers fully described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the invention, for instance,
the polynucleotides of the invention, may be used as a hybridization probe for RNA, cDNA and
genomic DNA to isolate full-length cDNAs and genomic clones encoding gidA1 and to isolate cDNA
and genomic clones of other genes that have a high identity, particularly high sequence identity, to the
gidA1 gene. Such probes generally will comprise at least 15 nucleotide residues or base pairs.
Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50
nucleotide residues or base pairs. Particularly preferred probes will have at least 20 nucleotide residues
or base pairs and will have lee than 30 nucleotide residues or base pairs.
A coding region of a gidA1 gene may be isolated by screening using a DNA sequence provided
in Table 1 [SEQ ID NO: 1 or 3] to synthesize an oligonucleotide probe. A labeled oligonucleotide
having a sequence complementary to that of a gene of the invention is then used to screen a library of
cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
There are several methods available and well known to those skilled in the art to obtain full-length
DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification
of cDNA ends (RACE) (see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988).
Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech
Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the
Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and
an 'adaptor' sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to
amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific
oligonucleotide primers. The PCR reaction is then repeated using "nested" primers, that is, primers
designed to anneal within the amplified product (typically an adaptor specific primer that anneals
further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected
gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full-length
DNA constructed either by joining the product directly to the existing DNA to give a
complete sequence, or carrying out a separate full-length PCR using the new sequence information
for the design of the 5' primer.
The polynucleotides and polypeptides of the invention may be employed, for example, as
research reagents and materials for discovery of treatments of and diagnostics for diseases, particularly
human diseases, as further discussed herein relating to polynucleotide assays.
The polynucleotides of the invention that are oligonucleotides derived from a sequence of
Table 1 [SEQ ID NOS:1 or 2 or 3 or 4] may be used in the processes herein as described, but
preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in
part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have
utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
The invention also provides polynucleotides that encode a polypeptide that is the mature
protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature
polypeptide (when the mature form has more than one polypeptide chain, for instance). Such
sequences may play a role in processing of a protein from precursor to a mature form, may allow
protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein
for assay or production, among other things. As generally is the case in vivo, the additional amino acids
may be processed away from the mature protein by cellular enzymes.
For each and every polynucleotide of the invention there is provided a polynucleotide
complementary to it. It is preferred that these complementary polynucleotides are fully complementary
to each polynucleotide with which they are complementary.
A precursor protein, having a mature form of the polypeptide fused to one or more
prosequences may be an inactive form of the polypeptide. When prosequences are removed such
inactive precursors generally are activated. Some or all of the prosequences may be removed before
activation. Generally, such precursors are called proproteins.
In addition to the standard A, G, C, T/U representations for nucleotides, the term "N" may
also be used in describing certain polynucleotides of the invention. "N" means that any of the four
DNA or RNA nucleotides may appear at such a designated position in the DNA or RNA sequence,
except it is preferred that N is not a nucleic acid that when taken in combination with adjacent
nucleotide positions, when read in the correct reading frame, would have the effect of generating a
premature termination codon in such reading frame.
In sum, a polynucleotide of the invention may encode a mature protein, a mature protein plus a
leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one
or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, which is a
precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are
removed during processing steps that produce active and mature forms of the polypeptide.
Vectors, Host Cells, Expression Systems
The invention also relates to vectors that comprise a polynucleotide or polynucleotides of the
invention, host cells that are genetically engineered with vectors of the invention and the production of
polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be
employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
Recombinant polypeptides of the present invention may be prepared by processes well known
in those skilled in the art from genetically engineered host cells comprising expression systems.
Accordingly, in a further aspect, the present invention relates to expression systems which comprise a
polynucleotide or polynucleotides of the present invention, to host cells which are genetically
engineered with such expression systems, and to the production of polypeptides of the invention by
recombinant techniques.
For recombinant production of the polypeptides of the invention, host cells can be genetically
engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
Introduction of a polynucleotide into the host cell can be effected by methods described in many
standard laboratory manuals, such as Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY,
(1986) and Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate
transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated
transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
Representative examples of appropriate hosts include bacterial cells, such as cells of
streptococci, staphylococci, enterococci E. coli, streptomyces, cyanobacteria, Bacillus subtilis, and
Staphylococcus aureus; fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a
basidiomycete, Candida albicans and Aspergillus; insect cells such as cells of Drosophila S2 and
Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes
melanoma cells; and plant cells, such as cells of a gymnosperm or angiosperm.
A great variety of expression systems can be used to produce the polypeptides of the invention.
Such vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example,
vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes,
from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova
viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses,
picornaviruses and retroviruses, and vectors derived from combinations thereof, such as those derived
from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression
system constructs may contain control regions that regulate as well as engender expression. Generally,
any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a
polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may
be inserted into the expression system by any of a variety of well-known and routine techniques, such
as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL, (supra).
In recombinant expression systems in eukaryotes, for secretion of a translated protein into the
lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment,
appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may
be endogenous to the polypeptide or they may be heterologous signals.
Polypeptides of the invention can be recovered and purified from recombinant cell cultures by
well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or
cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin
chromatography. Most preferably, high performance liquid chromatography is employed for
purification. Well known techniques for refolding protein may be employed to regenerate active
conformation when the polypeptide is denatured during isolation and or purification.
Diagnostic, Prognostic, Serotyping and Mutation Assays
This invention is also related to the use of gidA1polynucleotides and polypeptides of the
invention for use as diagnostic reagents. Detection of gidA1 polynucleotides and/or polypeptides in a
eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for
diagnosis of disease, staging of disease or response of an infectious organism to drugs. Eukaryotes,
particularly mammals, and especially humans, particularly those infected or suspected to be infected
with an organism comprising the gidA1gene or protein, may be detected at the nucleic acid or amino
acid level by a variety of well known techniques as well as by methods provided herein.
Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained
from a putatively infected and/or infected individual's bodily materials. Polynucleotides from any of
these sources, particularly DNA or RNA, may be used directly for detection or may be amplified
enzymatically by using PCR or any other amplification technique prior to analysis. RNA, particularly
mRNA, cDNA and genomic DNA may also be used in the same ways. Using amplification,
characterization of the species and strain of infectious or resident organism present in an individual,
may be made by an analysis of the genotype of a selected polynucleotide of the organism. Deletions
and insertions can be detected by a change in size of the amplified product in comparison to a genotype
of a reference sequence selected from a related organism, preferably a different species of the same
genus or a different strain of the same species. Point mutations can be identified by hybridizing
amplified DNA to labeled gidA1 polynucleotide sequences. Perfectly or significantly matched
sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase
or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures
or renaturation kinetics. Polynucleotide sequence differences may also be detected by alterations in the
electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This
may be carried out with or without denaturing agents. Polynucleotide differences may also be detected
by direct DNA or RNA sequencing. See, for example, Myers et al., Science, 230: 1242 (1985).
Sequence changes at specific locations also may be revealed by nuclease protection assays, such as
RNase, V1 and S1 protection assay or a chemical cleavage method. See, for example, Cotton et al.,
Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
In another embodiment, an array of oligonucleotides probes comprising gidA nucleotide
sequence or fragments thereof can be constructed to conduct efficient screening of, for example,
genetic mutations, serotype, taxonomic classification or identification. Array technology methods are
well known and have general applicability and can be used to address a variety of questions in
molecular genetics including gene expression, genetic linkage, and genetic variability (see, for
example, Chee et al., Science, 274: 610(1996)).
Thus in another aspect, the present invention relates to a diagnostic kit which comprises:
(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 1 or
3, or a fragment thereof; (b) a nucleotide sequence complementary to that of (a); (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or 4 or a
fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID
NO:2 or 4.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial
component. Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among
others.
This invention also relates to the use of polynucleotides of the present invention as diagnostic
reagents. Detection of a mutated form of a polynucleotide of the invention, preferable, SEQ ID NO: 1
or 3, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to,
or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of
disease, or a susceptibility to a disease, which results from under-expression, over-expression or altered
expression of the polynucleotide. Organisms, particularly infectious organisms, carrying mutations in
such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as
those described elsewhere herein.
The nucleotide sequences of the present invention are also valuable for organism chromosome
identification. The sequence is specifically targeted to, and can hybridize with, a particular location on
an organism's chromosome, particularly to a Staphylococcus aureus chromosome. The mapping of
relevant sequences to chromosomes according to the present invention may be an important step in
correlating those sequences with pathogenic potential and/or an ecological niche of an organism and/or
drug resistance of an organism, as well as the essentiality of the gene to the organism. Once a sequence
has been mapped to a precise chromosomal location, the physical position of the sequence on the
chromosome can be correlated with genetic map data. Such data may be found on-line in a sequence
database. The relationship between genes and diseases that have been mapped to the same
chromosomal region are then identified through known genetic methods, for example, through linkage
analysis (coinheritance of physically adjacent genes) or mating studies, such as by conjugation.
The differences in a polynucleotide and/or polypeptide sequence between organisms
possessing a first phenotype and organisms possessing a different, second different phenotype can
also be determined. If a mutation is observed in some or all organisms possessing the first
phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to
be the causative agent of the first phenotype.
Cells from an organism carrying mutations or polymorphisms (allelic variations) in a
polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or
polypeptide level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR
can be used to detect mutations in the RNA. It is particularly preferred to use RT-PCR in
conjunction with automated detection systems, such as, for example, GeneScan. RNA, cDNA or
genomic DNA may also be used for the same purpose, PCR. As an example, PCR primers
complementary to a polynucleotide encoding gidA1 polypeptide can be used to identify and analyze
mutations. Examples of representative primers are shown below in Table 2.
The invention also includes primers of the formula:
X-(R1)m-(R2)-(R3)n-Y
wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, and at
the 3' end of the molecule, Y is hydrogen, a metal or a modified nucleotide residue, R1 and R3 are any
nucleic acid residue or modified nucleotide residue, m is an integer between I and 20 or zero, n is an
integer between 1 and 20 or zero, and R2 is a primer sequence of the invention, particularly a primer
sequence selected from Table 2. In the polynucleotide formula above R2 is oriented so that its 5' end
nucleotide residue is at the left, bound to R1, and its 3' end nucleotide residue is at the right, bound to
R3. Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1,
may be either a heteropolymer or a homopolymer, preferably a heteropolymer being complementary to
a region of a polynucleotide of Table 1. In a preferred embodiment m and/or n is an integer between 1
and 10.
The invention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5'
and/or the 3' end. These primers may be used for, among other things, amplifying gidA1 DNA and/or
RNA isolated from a sample derived from an individual, such as a bodily material. The primers may be
used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may
then be subject to various techniques for elucidation of the polynucleotide sequence. In this way,
mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the
infection or its stage or course, or to serotype and/or classify the infectious agent.
The invention further provides a process for diagnosing, disease, preferably bacterial
infections, more preferably infections caused by Staphylococcus aureus, comprising determining from
a sample derived from an individual, such as a bodily material, an increased level of expression of
polynucleotide having a sequence of Table 1 [SEQ ID NO: 1 or 3]. Increased or decreased
expression of a gidA1 polynucleotide can be measured using any on of the methods well known in
the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR,
RNase protection, Northern blotting, spectrometry and other hybridization methods.
In addition, a diagnostic assay in accordance with the invention for detecting over-expression
of gidA1 polypeptide compared to normal control tissue samples may be used to detect the
presence of an infection, for example. Assay techniques that can be used to determine levels of a gidA1
polypeptide, in a sample derived from a host, such as a bodily material, are well-known to those of skill
in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot
analysis, antibody sandwich assays, antibody detection and ELISA assays.
Differential Expression
The polynucleotides and polynucleotides of the invention may be used as reagents for
differential screening methods. There are many differential screening and differential display methods
known in the art in which the polynucleotides and polypeptides of the invention may be used. For
example, the differential display technique is described by Chuang et al., J. Bacteriol. 175:2026-2036
(1993). This method identifies those genes which are expressed in an organism by identifying
mRNA present using randomly-primed RT-PCR. By comparing pre-infection and post infection
profiles, genes up and down regulated during infection can be identified and the RT-PCR product
sequenced and matched to ORF "unknowns."
In Vivo Expression Technology (IVET) is described by Camilli et al., Proc. Nat'l. Acad. Sci.
USA. 91:2634-2638 (1994). IVET identifies genes up-regulated during infection when compared to
laboratory cultivation, implying an important role in infection. ORFs identified by this technique are
implied to have a significant role in infection establishment and/or maintenance. In this technique
random chromosomal fragments of target organism are cloned upstream of a promoter-less
recombinase gene in a plasmid vector. This construct is introduced into the target organism which
carries an antibiotic resistance gene flanked by resolvase sites. Growth in the presence of the
antibiotic removes from the population those fragments cloned into the plasmid vector capable of
supporting transcription of the recombinase gene and therefore have caused loss of antibiotic
resistance. The resistant pool is introduced into a host and at various times after infection bacteria
may be recovered and assessed for the presence of antibiotic resistance. The chromosomal fragment
carried by each antibiotic sensitive bacterium should carry a promoter or portion of a gene normally
upregulated during infection. Sequencing upstream of the recombinase gene allows identification of
the up regulated gene.
RT-PCR may also be used to analyze gene expression patterns. For RT PCR using the
polynucleotides of the invention, messenger RNA is isolated from bacterial infected tissue, e.g., 48
hour murine lung infections, and the amount of each mRNA species assessed by reverse
transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene
specific primer pairs. The determination of the presence and amount of a particular mRNA species
by quantification of the resultant PCR product provides information on the bacterial genes which are
transcribed in the infected tissue. Analysis of gene transcription can be carried out at different times
of infection to gain a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a
clearer understanding of which gene products represent targets for screens for antibacterials.
Because of the gene specific nature of the PCR primers employed it should be understood that the
bacterial mRNA preparation need not be free of mammalian RNA. This allows the investigator to
carry out a simple and quick RNA preparation from infected tissue to obtain bacterial mRNA species
which are very short lived in the bacterium (in the order of 2 minute halflives). Optimally the
bacterial mRNA is prepared from infected murine lung tissue by mechanical disruption in the
presence of TRIzole (GIBCO-BRL) for very short periods of time, subsequent processing according
to the manufacturers of TRIzole reagent and DNAase treatment to remove contaminating DNA.
Preferably the process is optimized by finding those conditions which give a maximum amount of
Staphylococcus aureus 16S ribosomal RNA as detected by probing Northerns with a suitably labeled
sequence specific oligonucleotide probe. Typically a 5' dye labeled primer is used in each PCR
primer pair in a PCR reaction which is terminated optimally between 8 and 25 cycles. The PCR
products are separated on 6% polyacrylamide gels with detection and quantification using
Gene Scanner (manufactured by ABI).
Gridding and Polynucleotide Subtraction
Methods have been described for obtaining information about gene expression and identity
using so called "high density DNA arrays" or grids. See, e.g., M. Chee et al., Science, 274:610-614
(1996) and other references cited therein. Such gridding assays have been employed to
identify certain novel gene sequences, referred to as Expressed Sequence Tags (EST) (Adams et a.,
Science, 252:1651-1656 (1991)). A variety of techniques have also been described for identifying
particular gene sequences on the basis of their gene products. For example, see International
Patent Application No. WO91/07087, published May 30, 1991. In addition, methods have been
described for the amplification of desired sequences. For example, see International Patent
Application No. WO91/17271, published November 14, 1991.
The polynucleotides of the invention may be used as components of polynucleotide arrays,
preferably high density arrays or grids. These high density arrays are particularly useful for
diagnostic and prognostic purposes. For example, a set of spots each comprising a different gene,
and further comprising a polynucleotide or polynucleotides of the invention, may be used for
probing, such as using hybridization or nucleic acid amplification, using a probes obtained or
derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or
related sequence in an individual. Such a presence may indicate the presence of a pathogen,
particularly Staphylococcus aureus, and may be useful in diagnosing and/or prognosing disease or
a course of disease. A grid comprising a number of variants of the polynucleotide sequence of
SEQ ID NO:1 or 3 are preferred. Also preferred is a comprising a number of variants of a
polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO:2 or 4.
Antibodies
The polypeptides and polynucleotides of the invention or variants thereof, or cells expressing
the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or
polynucleotides respectively.
In certain preferred embodiments of the invention there are provided antibodies against gidA1
polypeptides or polynucleotides.
Antibodies generated against the polypeptides or polynucleotides of the invention can be
obtained by administering the polypeptides and/or polynucleotides of the invention, or epitope-bearing
fragments of either or both, analogues of either or both, or cells expressing either or both, to an animal,
preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any
technique known in the art that provides antibodies produced by continuous cell line cultures can be
used. Examples include various techniques, such as those in Kohler, G. and Milstein, C., Nature 256:
495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be
adapted to produce single chain antibodies to polypeptides or polynucleotides of this invention. Also,
transgenic mice, or other organisms such as other mammals, may be used to express humanized
antibodies immunospecific to the polypeptides or polynucleotides of the invention.
Alternatively, phage display technology may be utilized to select antibody genes with
binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes
of lymphocytes from humans screened for possessing anti-gidA1 or from naive libraries
(McCafferty, et al., (1990), Nature 348, 552-554; Marks, et al., (1992) Biotechnology 10, 779-783).
The affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et
al., (1991) Nature 352: 628).
The above-described antibodies may be employed to isolate or to identify clones expressing the
polypeptides or polynucleotides of the invention to purify the polypeptides or polynucleotides by, for
example, affinity chromatography.
Thus, among others, antibodies against gidA1-polypeptide or gidA1-polynudeotide may be
employed to treat infections, particularly bacterial infections.
Polypeptide variants include antigenically, epitopically or immunologically equivalent
variants form a particular aspect of this invention.
A polypeptide or polynucleotide of the invention, such as an antigenically or
immunologically equivalent derivative or a fusion protein of the polypeptide is used as an antigen to
immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stability
to the polypeptide. The antigen may be associated, for example by conjugation, with an
immunogenic carrier protein for example bovine serum albumin, keyhole limpet haemocyanin or
tetanus toxoid. Alternatively, a multiple antigenic polypeptide comprising multiple copies of the
polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be
sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
Preferably, the antibody or variant thereof is modified to make it less immunogenic in the
individual. For example, if the individual is human the antibody may most preferably be
"humanized," where the complimentarity determining region or regions of the hybridoma-derived
antibody has been transplanted into a human monoclonal antibody, for example as described in Jones
et al. (1986), Nature 321, 522-525 or Tempest et al., (1991) Biotechnology 9, 266-273.
In accordance with an aspect of the invention, there is provided the use of a polynucleotide
of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
Among the particularly preferred embodiments of the invention are naturally occurring allelic variants
of gidA1 polynucleotides and polypeptides encoded thereby.
The use of a polynucleotide of the invention in genetic immunization will preferably employ
a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al.. Hum
Mol Genet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419), delivery of DNA
complexed with specific protein carriers (Wu et al., J Biol Chem. (1989) 264: 16985),
coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS USA, (1986) 83:
9551), encapsulation of DNA in various forms of liposomes (Kaneda et al., Science (1989) 243:
375), particle bombardment (Tang et al., Nature (1992) 356:152, Eisenbraun et al., DNA Cell Biol
(1993) 12: 791) and in vivo infection using cloned retroviral vectors (Seeger et al., PNAS USA
(1984) 81: 5849).
Antagonists and Agonists - Assays and Molecules
Polypeptides and polynucleotides of the invention may also be used to assess the binding of
small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries,
and natural product mixtures. These substrates and ligands may be natural substrates and ligands or
may be structural or functional mimetics. See, e.g., Coligan et al., Current Protocols in Immunology
1(2): Chapter 5 (1991).
Polypeptides and polynucleotides of the present invention are responsible for many biological
functions, including many disease states, in particular the Diseases hereinbefore mentioned. It is
therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit
the function of the polypeptide or polynucleotide. Accordingly, in a further aspect, the present
invention provides for a method of screening compounds to identify those which stimulate or which
inhibit the function of a polypeptide or polynucleotide of the invention, as well as related polypeptides
and polynucleotides. In general, agonists or antagonists may be employed for therapeutic and
prophylactic purposes for such Diseases as hereinbefore mentioned. Compounds may be identified
from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural
product mixtures. Such agonists, antagonists or inhibitors so-identified may be natural or modified
substrates, ligands, receptors, enzymes, etc., as the case may be, of gidA I polypeptides and
polynucleotides; or may be structural or functional mimetics thereof (see Coligan et al., Current
Protocols in Immunology 1(2):Chapter 5 (1991)).
The screening methods may simply measure the binding of a candidate compound to the
polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide,
or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the
candidate compound. Alternatively, the screening method may involve competition with a labeled
competitor. Further, these screening methods may test whether the candidate compound results in a
signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection
systems appropriate to the cells comprising the polypeptide or polynucleotide. Inhibitors of
activation are generally assayed in the presence of a known agonist and the effect on activation by
the agonist by the presence of the candidate compound is observed. Constitutively active
polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in
screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by
testing whether the candidate compound results in inhibition of activation of the polypeptide or
polynucleotide, as the case may be. Further, the screening methods may simply comprise the steps
of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the
present invention, to form a mixture, measuring gidA1 polypeptide and/or polynucleotide activity in
the mixture, and comparing the gidA1 polypeptide and/or polynucleotide activity of the mixture to a
standard. Fusion proteins, such as those made from Fc portion and gidA1 polypeptide, as
hereinbefore described, can also be used for high-throughput screening assays to identify antagonists
of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally
related polypeptides (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et
al., J Biol Chem, 270(16):9459-9471 (1995)).
The polynucleotides, polypeptides and antibodies that bind to and/or interact with a
polypeptide of the present invention may also be used to configure screening methods for detecting
the effect of added compounds on the production of mRNA and/or polypeptide in cells. For
example, an ELISA assay may be constructed for measuring secreted or cell associated levels of
polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This
can be used to discover agents which may inhibit or enhance the production of polypeptide (also
called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
The invention also provides a method of screening compounds to identify those which enhance
(agonist) or block (antagonist) the action of gidA1 polypeptides or polynucleotides, particularly those
compounds that are bacteristatic and/or bactericidal. The method of screening may involve high-throughput
techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a
cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof,
comprising gidA1polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the
absence or the presence of a candidate molecule that may be a gidA1 agonist or antagonist. The ability
of the candidate molecule to agonize or antagonize the gidA1 polypeptide is reflected in decreased
binding of the labeled ligand or decreased production of product from such substrate. Molecules that
bind gratuitously, i.e., without inducing the effects of gidA1 polypeptide are most likely to be good
antagonists. Molecules that bind well and, as the case may be, increase the rate of product production
from substrate, increase signal transduction, or increase chemical channel activity are agonists.
Detection of the rate or level of, as the case may be, production of product from substrate, signal
transduction, or chemical channel activity may be enhanced by using a reporter system. Reporter
systems that may be useful in this regard include but are not limited to colorimetric, labeled substrate
converted into product, a reporter gene that is responsive to changes in gidA1polynucleotide or
polypeptide activity, and binding assays known in the art.
Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if
any, for such polypeptide, through standard receptor binding techniques known in the art. These
techniques include, but are not limited to, ligand binding and crosslinking assays in which the
polypeptide is labeled with a radioactive isotope (for instance, 125I), chemically modified (for
instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and
incubated with a source of the putative receptor (e.g., cells, cell membranes, cell supernatants,
tissue extracts, bodily materials). Other methods include biophysical techniques such as surface
plasmon resonance and spectroscopy. These screening methods may also be used to identify
agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to
its receptor(s), if any. Standard methods for conducting such assays are well understood in the art.
The fluorescence polarization value for a fluorescently-tagged molecule depends on the
rotational correlation time or tumbling rate. Protein complexes, such as formed by gidA1
polypeptide associating with another gidA1 polypeptide or other polypeptide, labeled to comprise
a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled
monomeric protein. It is preferred that this method be used to characterize small molecules that
disrupt polypeptide complexes.
Fluorescence energy transfer may also be used characterize small molecules that interfere
with the formation of gidA1 polypeptide dimers, trimers, tetramers or higher order structures, or
structures formed by gidA1 polypeptide bound to another polypeptide. gidA1 polypeptide can be
labeled with both a donor and acceptor fluorophore. Upon mixing of the two labeled species and
excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing
fluorescence of the acceptor. Compounds that block dimerization will inhibit fluorescence energy
transfer.
Surface plasmon resonance can be used to monitor the effect of small molecules on gidA1
polypeptide self-association as well as an association of gidA1 polypeptide and another
polypeptide or small molecule. gidA1 polypeptide can be coupled to a sensor chip at low site
density such that covalently bound molecules will be monomeric. Solution protein can then
passed over the gidA1 polypeptide -coated surface and specific binding can be detected in real-time
by monitoring the change in resonance angle caused by a change in local refractive index.
This technique can be used to characterize the effect of small molecules on kinetic rates and
equilibrium binding constants for gidA1 polypeptide self-association as well as an association of
gidA1 polypeptide and another polypeptide or small molecule.
A scintillation proximity assay may be used to characterize the interaction between an
association of gidA1 polypeptide with another gidA1 polypeptide or a different polypeptide .
gidA1 polypeptide can be coupled to a scintillation-filled bead. Addition of radio-labeled gidA1
polypeptide results in binding where the radioactive source molecule is in close proximity to the
scintillation fluid. Thus, signal is emitted upon gidA1 polypeptide binding and compounds that
prevent gidA1 polypeptide self-association or an association of gidA1 polypeptide and another
polypeptide or small molecule will diminish signal.
ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology
Research Institute). They couple the self-association of macromolecules to the closing of
gramacidin-facilitated ion channels in suspended membrane bilayers and hence to a measurable
change in the admittance (similar to impedence) of the biosensor. This approach is linear over six
decades of admittance change and is ideally suited for large scale, high through-put screening of
small molecule combinatorial libraries.
In other embodiments of the invention there are provided methods for identifying compounds
which bind to or otherwise interact with and inhibit or activate an activity or expression of a
polypeptide and/or polynucleotide of the invention comprising: contacting a polypeptide and/or
polynucleotide of the invention with a compound to be screened under conditions to permit binding to
or other interaction between the compound and the polypeptide and/or polynucleotide to assess the
binding to or other interaction with the compound, such binding or interaction preferably being
associated with a second component capable of providing a detectable signal in response to the binding
or interaction of the polypeptide and/or polynucleotide with the compound; and determining whether
the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of
the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from
the binding or interaction of the compound with the polypeptide and/or polynucleotide.
Another example of an assay for gidA1 agonists is a competitive assay that combines gidA1
and a potential agonist with gidA1-binding molecules, recombinant gidA1 binding molecules, natural
substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive
inhibition assay. gidA1 can be labeled, such as by radioactivity or a colorimetric compound, such that
the number of gidA1 molecules bound to a binding molecule or converted to product can be determined
accurately to assess the effectiveness of the potential antagonist.
Potential antagonists include, among others, small organic molecules, peptides, polypeptides
and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or
extinguish its activity or expression. Potential antagonists also may be small organic molecules, a
peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a
binding molecule, such as a binding molecule, without inducing gidA1-induced activities, thereby
preventing the action or expression of gidA1 polypeptides and/or polynucleotides by excluding gidA1
polypeptides and/or polynucleotides from binding.
Potential antagonists include a small molecule that binds to and occupies the binding site of the
polypeptide thereby preventing binding to cellular binding molecules, such that normal biological
activity is prevented. Examples of small molecules include but are not limited to small organic
molecules, peptides or peptide-like molecules. Other potential antagonists include antisense molecules
(see Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE
INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these
molecules). Preferred potential antagonists include compounds related to and variants of gidA1.
Other examples of potential polypeptide antagonists include antibodies or, in some cases,
oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes,
etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors,
enzymes, etc.; or small molecules which bind to the polypeptide of the present invention but do not
elicit a response, so that the activity of the polypeptide is prevented.
Certain of the polypeptides of the invention are biomimetics, functional mimetics of the natural
gidA1 polypeptide. These functional mimetics may be used for, among other things, antagonizing the
activity of gidA1 polypeptide or as a antigen or immunogen in a manner described elsewhere herein.
Functional mimetics of the polypeptides of the invention include but are not limited to truncated
polypeptides. For example, preferred functional mimetics include, a polypeptide comprising the
polypeptide sequence set forth in SEQ ID NO:2 lacking 20, 30, 40, 50, 60, 70 or 80 amino- or carboxy-terminal
amino acid residues, including fusion proteins comprising one or more of these truncated
sequences. Polynucleotides encoding each of these functional mimetics may be used as expression
cassettes to express each mimetic polypeptide. It is preferred that these cassettes comprise 5'and 3'
restriction sites to allow for a convenient means to ligate the cassettes together when desired. It is
further preferred that these cassettes comprise gene expression signals known in the art or described
elsewhere herein.
Thus, in another aspect, the present invention relates to a screening kit for identifying
agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for a polypeptide and/or
polynucleotide of the present invention; or compounds which decrease or enhance the production of
such polypeptides and/or polynucleotides, which comprises:
(a) a polypeptide and/or a polynucleotide of the present invention; (b) a recombinant cell expressing a polypeptide and/or polynucleotide of the present invention; (c) a cell membrane expressing a polypeptide and/or polynucleotide of the present invention; or (d) antibody to a polypeptide and/or polynucleotide of the present invention;
which polypeptide is preferably that of SEQ ID NO:2, and which polynucleotide is preferably that of
SEQ ID NO:1.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial
component.
It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide
of the present invention may also be used in a method for the structure-based design of an agonist,
antagonist or inhibitor of the polypeptide and/or polynucleotide, by:
(a) determining in the first instance the three-dimensional structure of the polypeptide and/or
polynucleotide, or complexes thereof; (b) deducing the three-dimensional structure for the likely reactive site(s), binding site(s) or motif(s)
of an agonist, antagonist or inhibitor; (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced
binding site(s), reactive site(s), and/or motif(s); and (d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors.
It will be further appreciated that this will normally be an iterative process, and this iterative process
may be performed using automated and computer-controlled steps.
In a further aspect, the present invention provides methods of treating abnormal conditions
such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated
activity of, or a decreased activity of gidA1 polypeptide and/or polynucleotide.
If the expression and/or activity of the polypeptide and/or polynucleotide is in excess, several
approaches are available. One approach comprises administering to an individual in need thereof an
inhibitor compound (antagonist) as herein described, optionally in combination with a pharmaceutically
acceptable carrier, in an amount effective to inhibit the function and/or expression of the polypeptide
and/or polynucleotide, such as, for example, by blocking the binding of ligands, substrates, receptors,
enzymes, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition. In
another approach, soluble forms of the polypeptides still capable of binding the ligand, substrate,
enzymes, receptors, etc. in competition with endogenous polypeptide and/or polynucleotide may be
administered. Typical examples of such competitors include fragments of the gidA1 polypeptide
and/or polypeptide.
In a further aspect, the present invention relates to genetically engineered soluble fusion
proteins comprising a polypeptide of the present invention, or a fragment thereof, and various
portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses
(IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part of the heavy chain of
human IgG, particularly IgG1, where fusion takes place at the hinge region. In a particular
embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can
be cleaved with blood clotting factor Xa. Furthermore, this invention relates to processes for the
preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening,
diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding
such fusion proteins. Examples of fusion protein technology can be found in International Patent
Application Nos. WO94/29458 and WO94/22914.
In still another approach, expression of the gene encoding endogenous gidA1 polypeptide
can be inhibited using expression blocking techniques. This blocking may be targeted against any
step in gene expression, but is preferably targeted against transcription and/or translation. An
examples of a known technique of this sort involve the use of antisense sequences, either internally
generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56:560 in
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL
(1988)). Alternatively, oligonucleotides which form triple helices with the gene can be supplied
(see, for example, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)
241:456; Dervan et al., Science (1991) 251:1360). These oligomers can be administered per se or
the relevant oligomers can be expressed in vivo.
Each of the polynucleotide sequences provided herein may be used in the discovery and
development of antibacterial compounds. The encoded protein, upon expression, can be used as a
target for the screening of antibacterial drugs. Additionally, the polynucleotide sequences encoding
the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating
sequences of the respective mRNA can be used to construct antisense sequences to control the
expression of the coding sequence of interest.
The invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist
of the invention to interfere with the initial physical interaction between a pathogen or pathogens and
a eukaryotic, preferably mammalian, host responsible for sequelae of infection. In particular, the
molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram
positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix
proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block bacterial
adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial
gidA1 proteins that mediate tissue damage and/or; to block the normal progression of pathogenesis in
infections initiated other than by the implantation of in-dwelling devices or by other surgical
techniques.
In accordance with yet another aspect of the invention, there are provided gidA1 agonists and
antagonists, preferably bacteristatic or bactericidal agonists and antagonists.
The antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit
and/or treat diseases.
Helicobacter pylori (herein "H. pylori") bacteria infect the stomachs of over one-third of the
world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research
on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for
Research on Cancer, Lyon, France, http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the
International Agency for Research on Cancer recently recognized a cause-and-effect relationship
between H. pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite)
carcinogen. Preferred antimicrobial compounds of the invention (agonists and antagonists of gidA1
polypeptides and/or polynucleotides) found using screens provided by the invention, or known in the
art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H. pylori infection.
Such treatment should decrease the advent of H. pylori-induced cancers, such as gastrointestinal
carcinoma. Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis.
Vaccines
There are provided by the invention, products, compositions and methods for assessing gidA1
expression, treating disease, assaying genetic variation, and administering a gidA1 polypeptide and/or
polynucleotide to an organism to raise an immunological response against a bacteria, especially a
Staphylococcus aureus bacteria.
Another aspect of the invention relates to a method for inducing an immunological response
in an individual, particularly a mammal which comprises inoculating the individual with gidA1
polynucleotide and/or polypeptide, or a fragment or variant thereof, adequate to produce antibody
and/ or T cell immune response to protect said individual from infection, particularly bacterial
infection and most particularly Staphylococcus aureus infection. Also provided are methods
whereby such immunological response slows bacterial replication. Yet another aspect of the
invention relates to a method of inducing immunological response in an individual which comprises
delivering to such individual a nucleic acid vector, sequence or ribozyme to direct expression of
gidA1 polynucleotide and/or polypeptide, or a fragment or a variant thereof, for expressing gidA1
polynucleotide and/or polypeptide, or a fragment or a variant thereof in vivo in order to induce an
immunological response, such as, to produce antibody and/ or T cell immune response, including, for
example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a
human, from disease, whether that disease is already established within the individual or not. One
example of administering the gene is by accelerating it into the desired cells as a coating on particles
or otherwise. Such nucleic acid vector may comprise DNA, RNA, a ribozyme, a modified nucleic
acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-protein complex.
A further aspect of the invention relates to an immunological composition that when
introduced into an individual, preferably a human, capable of having induced within it an
immunological response, induces an immunological response in such individual to a gidA1
polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a
recombinant gidA1 polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA
and/or RNA which encodes and expresses an antigen of said gidA1 polynucleotide, polypeptide
encoded therefrom, or other polypeptide of the invention. The immunological response may be used
therapeutically or prophylactically and may take the form of antibody immunity and/or cellular
immunity, such as cellular immunity arising from CTL or CD4+ T cells.
A gidA1 polypeptide or a fragment thereof may be fused with co-protein or chemical moiety
which may or may not by itself produce antibodies, but which is capable of stabilizing the first
protein and producing a fused or modified protein which will have antigenic and/or immunogenic
properties, and preferably protective properties. Thus fused recombinant protein, preferably further
comprises an antigenic co-protein, such as lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase
(GST) or beta-galactosidase, or any other relatively large co-protein which solubilizes
the protein and facilitates production and purification thereof. Moreover, the co-protein may act as
an adjuvant in the sense of providing a generalized stimulation of the immune system of the
organism receiving the protein. The co-protein may be attached to either the amino- or carboxy-terminus
of the first protein.
Provided by this invention are compositions, particularly vaccine compositions, and methods
comprising the polypeptides and/or polynucleotides of the invention and immunostimulatory DNA
sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).
Also, provided by this invention are methods using the described polynucleotide or
particular fragments thereof, which have been shown to encode non-variable regions of bacterial cell
surface proteins, in polynucleotide constructs used in such genetic immunization experiments in
animal models of infection with Staphylococcus aureus. Such experiments will be particularly useful
for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is
believed that this approach will allow for the subsequent preparation of monoclonal antibodies of
particular value, derived from the requisite organ of the animal successfully resisting or clearing
infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection,
particularly Staphylococcus aureus infection, in mammals, particularly humans.
A polypeptide of the invention may be used as an antigen for vaccination of a host to
produce specific antibodies which protect against invasion of bacteria, for example by blocking
adherence of bacteria to damaged tissue. Examples of tissue damage include wounds in skin or
connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by
implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat,
mammary glands, urethra or vagina.
The invention also includes a vaccine formulation which comprises an immunogenic
recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such
as a pharmaceutically acceptable carrier. Since the polypeptides and polynucleotides may be broken
down in the stomach, each is preferably administered parenterally, including, for example,
administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations
suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions
which may contain anti-oxidants, buffers, bacteristatic compounds and solutes which render the
formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and
non-aqueous sterile suspensions which may include suspending agents or thickening agents. The
formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules
and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile
liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems
for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems
known in the art. The dosage will depend on the specific activity of the vaccine and can be readily
determined by routine experimentation.
While the invention has been described with reference to certain gidA1 polypeptides and
polynucleotides, it is to be understood that this covers fragments of the naturally occurring
polypeptides and polynucleotides, and similar polypeptides and polynucleotides with additions,
deletions or substitutions which do not substantially affect the immunogenic properties of the
recombinant polypeptides or polynucleotides.
Compositions, kits and administration
In a further aspect of the invention there are provided compositions comprising a gidA1
polynucleotide and/or a gidA1 polypeptide for administration to a cell or to a multicellular organism.
The invention also relates to compositions comprising a polynucleotide and/or a polypeptides
discussed herein or their agonists or antagonists. The polypeptides and polynucleotides of the
invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with
cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to an individual.
Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a
polypeptide and/or polynucleotide of the invention and a pharmaceutically acceptable carrier or
excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water,
glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration.
The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more
containers filled with one or more of the ingredients of the aforementioned compositions of the
invention.
Polypeptides, polynucleotides and other compounds of the invention may be employed alone or
in conjunction with other compounds, such as therapeutic compounds.
The pharmaceutical compositions may be administered in any effective, convenient manner
including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal,
intramuscular, subcutaneous, intranasal or intradermal routes among others.
In therapy or as a prophylactic, the active agent may be administered to an individual as an
injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
Alternatively the composition may be formulated for topical application
for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops,
mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate
conventional additives, including, for example, preservatives, solvents to assist drug penetration, and
emollients in ointments and creams. Such topical formulations may also contain compatible
conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions.
Such carriers may constitute from about 1% to about 98% by weight of the formulation; more
usually they will constitute up to about 80% by weight of the formulation.
In a further aspect, the present invention provides for pharmaceutical compositions comprising
a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a
polypeptide and/or polynucleotide of the present invention, agonist or antagonist peptide or small
molecule compound, in combination with a pharmaceutically acceptable carrier or excipient. Such
carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. The invention further relates to pharmaceutical packs and kits comprising one or
more containers filled with one or more of the ingredients of the aforementioned compositions of the
invention. Polypeptides, polynucleotides and other compounds of the present invention may be
employed alone or in conjunction with other compounds, such as therapeutic compounds.
The composition will be adapted to the route of administration, for instance by a systemic or an
oral route. Preferred forms of systemic administration include injection, typically by intravenous
injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used.
Alternative means for systemic administration include transmucosal and transdermal administration
using penetrants such as bile salts or fusidic acids or other detergents. In addition, if a polypeptide or
other compounds of the present invention can be formulated in an enteric or an encapsulated
formulation, oral administration may also be possible. Administration of these compounds may also be
topical and/or localized, in the form of salves, pastes, gels, and the like.
For administration to mammals, and particularly humans, it is expected that the daily dosage
level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The
physician in any event will determine the actual dosage which will be most suitable for an individual
and will vary with the age, weight and response of the particular individual. The above dosages are
exemplary of the average case. There can, of course, be individual instances where higher or lower
dosage ranges are merited, and such are within the scope of this invention.
In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices
that are introduced to the body of an individual and remain in position for an extended time. Such
devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular
catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis
(CAPD) catheters.
The composition of the invention may be administered by injection to achieve a systemic
effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be
continued after surgery during the in-body time of the device. In addition, the composition could also
be used to broaden perioperative cover for any surgical technique to prevent bacterial wound
infections, especially Staphylococcus aureus wound infections.
Many orthopedic surgeons consider that humans with prosthetic joints should be considered
for antibiotic prophylaxis before dental treatment that could produce a bacteremia. Late deep
infection is a serious complication sometimes leading to loss of the prosthetic joint and is
accompanied by significant morbidity and mortality. It may therefore be possible to extend the use
of the active agent as a replacement for prophylactic antibiotics in this situation.
In addition to the therapy described above, the compositions of this invention may be used
generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in
wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction
with, antibiotic prophylaxis.
Alternatively, the composition of the invention may be used to bathe an indwelling device
immediately before insertion. The active agent will preferably be present at a concentration of
1µg/ml to 10mg/ml for bathing of wounds or indwelling devices.
A vaccine composition is conveniently in injectable form. Conventional adjuvants may be
employed to enhance the immune response. A suitable unit dose for vaccination is 0.5-5
microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of
1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the
compounds of the invention which would preclude their administration to suitable individuals.
Sequence Databases, Sequences in a Tangible Medium, and Algorithms
Polynucleotide and polypeptide sequences form a valuable information resource with which to
determine their 2- and 3-dimensional structures as well as to identify further sequences of similar
homology. These approaches are most easily facilitated by storing the sequence in a computer
readable medium and then using the stored data in a known macromolecular structure program or to
search a sequence database using well known searching tools, such as GCC.
The polynucleotide and polypeptide sequences of the invention are particularly useful as
components in databases useful for search analyses as well as in sequence analysis algorithms. As
used in this section entitled "Sequence Databases, Sequences in a Tangible Medium, and
Algorithms," and in claims related to this section, the terms "polynucleotide of the invention" and
"polynucleotide sequence of the invention" mean any detectable chemical or physical characteristic
of a polynucleotide of the invention that is or may be reduced to or stored in a tangible medium,
preferably a computer readable form. For example, chromatographic scan data or peak data,
photographic data or scan data therefrom, called bases, and mass spectrographic data. As used in
this section entitled Databases and Algorithms and in claims related thereto, the terms "polypeptide
of the invention" and "polypeptide sequence of the invention" mean any detectable chemical or
physical characteristic of a polypeptide of the invention that is or may be reduced to or stored in a
tangible medium, preferably a computer readable form. For example, chromatographic scan data or
peak data, photographic data or scan data therefrom, and mass spectrographic data.
The invention provides a computer readable medium having stored thereon polypeptide
sequences of the invention and/or polynucleotide sequences of the invention. For example, a
computer readable medium is provided comprising and having stored thereon a member selected
from the group consisting of: a polynucleotide comprising the sequence of a polynucleotide of the
invention; a polypeptide comprising the sequence of a polypeptide sequence of the invention; a set of
polynucleotide sequences wherein at least one of the sequences comprises the sequence of a
polynucleotide sequence of the invention; a set of polypeptide sequences wherein at least one of the
sequences comprises the sequence of a polypeptide sequence of the invention; a data set representing
a polynucleotide sequence comprising the sequence of polynucleotide sequence of the invention; a
data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the
sequence of a polypeptide sequence of the invention; a polynucleotide comprising the sequence of a
polynucleotide sequence of the invention; a polypeptide comprising the sequence of a polypeptide
sequence of the invention; a set of polynucleotide sequences wherein at least one of the sequences
comprises the sequence of a polynucleotide sequence of the invention; a set of polypeptide
sequences wherein at least one of said sequences comprises the sequence of a polypeptide sequence
of the invention; a data set representing a polynucleotide sequence comprising the sequence of a
polynucleotide sequence of the invention; a data set representing a polynucleotide sequence
encoding a polypeptide sequence comprising the sequence of a polypeptide sequence of the
invention. The computer readable medium can be any composition of matter used to store
information or data, including, for example, commercially available floppy disks, tapes, chips, hard
drives, compact disks, and video disks.
Also provided by the invention are methods for the analysis of character sequences or
strings, particularly genetic sequences or encoded genetic sequences. Preferred methods of sequence
analysis include, for example, methods of sequence homology analysis, such as identity and
similarity analysis, RNA structure analysis, sequence assembly, cladistic analysis, sequence motif
analysis, open reading frame determination, nucleic acid base calling, nucleic acid base trimming,
and sequencing chromatogram peak analysis.
A computer based method is provided for performing homology identification. This method
comprises the steps of providing a first polynucleotide sequence comprising the sequence a
polynucleotide of the invention in a computer readable medium; and comparing said first
polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to identify
homology.
A computer based method is also provided for performing homology identification, said
method comprising the steps of: providing a first polypeptide sequence comprising the sequence of a
polypeptide of the invention in a computer readable medium; and comparing said first polypeptide
sequence to at least one second polynucleotide or polypeptide sequence to identify homology.
A computer based method is still further provided for polynucleotide assembly, said method
comprising the steps of: providing a first polynucleotide sequence comprising the sequence of a
polynucleotide of the invention in a computer readable medium; and screening for at least one
overlapping region between said first polynucleotide sequence and at least one second
polynucleotide or polypeptide sequence.
A computer based method is still further provided for polynucleotide assembly, said method
comprising the steps of: providing a first polypeptide sequence comprising a polypeptide of the
invention in a computer readable medium; and screening for at least one overlapping region between
said first polypeptide sequence and at least one second polynucleotide or polypeptide sequence.
In another preferred embodiment of the invention there is provided a computer readable
medium having stored thereon a member selected from the group consisting of: a polynucleotide
comprising the sequence of SEQ ID NO. 1 or 3; a polypeptide comprising the sequence of SEQ ID
NO. 2 or 4; a set of polynucleotide sequences wherein at least one of said sequences comprises the
sequence of SEQ ID NO. 1 or 3; a set of polypeptide sequences wherein at least one of said
sequences comprises the sequence of SEQ ID NO. 2 or 4; a data set representing a polynucleotide
sequence comprising the sequence of SEQ ID NO. 1 or 3; a data set representing a polynucleotide
sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO. 2 or 4; a
polynucleotide comprising the sequence of SEQ ID NO. 1 or 3; a polypeptide comprising the
sequence of SEQ ID NO. 2 or 4; a set of polynucleotide sequences wherein at least one of said
sequences comprises the sequence of SEQ ID NO. 1 or 3; a set of polypeptide sequences wherein at
least one of said sequences comprises the sequence of SEQ ID NO. 2 or 4; a data set representing a
polynucleotide sequence comprising the sequence of SEQ ID NO. 1 or 3; a data set representing a
polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO.
2 or 4. A further preferred embodiment of the invention provides a computer based method for
performing homology identification, said method comprising the steps of providing a polynucleotide
sequence comprising the sequence of SEQ ID NO. 1 or 3 in a computer readable medium; and
comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to
identify homology.
A still further preferred embodiment of the invention provides a computer based method for
performing homology identification, said method comprising the steps of: providing a polypeptide
sequence comprising the sequence of SEQ ID NO. 2 or 4 in a computer readable medium; and
comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to
identify homology.
A further embodiment of the invention provides a computer based method for
polynucleotide assembly, said method comprising the steps of: providing a first polynucleotide
sequence comprising the sequence of SEQ ID NO. 1 or 3 in a computer readable medium; and
screening for at least one overlapping region between said first polynucleotide sequence and a
second polynucleotide sequence.
A further embodiment of the invention provides a computer based method for performing
homology identification, said method comprising the steps of: providing a polynucleotide sequence
comprising the sequence of SEQ ID NO. 1 or 3 in a computer readable medium; and comparing said
polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify
homology.
All publications and references, including but not limited to patents and patent applications,
cited in this specification are herein incorporated by reference in their entirety as if each individual
publication or reference were specifically and individually indicated to be incorporated by reference
herein as being fully set forth. Any patent application to which this application claims priority is
also incorporated by reference herein in its entirety in the manner described above for publications
and references.
GLOSSARY
The following definitions are provided to facilitate understanding of certain terms used
frequently herein.
"Antibody(ies)" as used herein includes polyclonal and monoclonal antibodies, chimeric,
single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab
or other immunoglobulin expression library.
"Antigenically equivalent derivative(s)" as used herein encompasses a polypeptide,
polynucleotide, or the equivalent of either which will be specifically recognized by certain
antibodies which, when raised to the protein, polypeptide or polynucleotide according to the
invention, interferes with the immediate physical interaction between pathogen and mammalian host.
"Bispecific antibody(ies)" means an antibody comprising at least two antigen binding
domains, each domain directed against a different epitope.
"Bodily material(s) means any material derived from an individual or from an organism
infecting, infesting or inhabiting an individual, including but not limited to, cells, tissues and waste,
such as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin,
urine, stool or autopsy materials..
"Disease(s)" means any disease caused by or related to infection by a bacteria, including, for
example, disease, such as, infections of the upper respiratory tract (e.g., otitis media, bacterial tracheitis,
acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective
endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess), CNS
(e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and
orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, intrarenal and perinephric
absces, toxic shock syndrome), skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis, wound
infection, bacterial myositis) bone and joint (e.g., septic arthritis, osteomyelitis).
"Fusion protein(s)" refers to a protein encoded by two, often unrelated, fused genes or
fragments thereof. In one example, EP-A-0464 discloses fusion proteins comprising various portions
of constant region of immunoglobulin molecules together with another human protein or part
thereof. In many cases, employing an immunoglobulin Fc region as a part of a fusion protein is
advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic
properties [see, e.g., EP-A 0232262]. On the other hand, for some uses it would be desirable to be
able to delete the Fc part after the fusion protein has been expressed, detected and purified.
"Host cell(s)" is a cell which has been transformed or transfected, or is capable of
transformation or transfection by an exogenous polynucleotide sequence.
"Identity," as known in the art, is a relationship between two or more polypeptide sequences or
two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences.
In the art, "identity" also means the degree of sequence relatedness between polypeptide or
polynucleotide sequences, as the case may be, as determined by the match between strings of such
sequences. "Identity" can be readily calculated by known methods, including but not limited to those
described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New
York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press,
New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G.,
Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M
Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073
(1988). Methods to determine identity are designed to give the largest match between the sequences
tested. Moreover, methods to determine identity are codified in publicly available computer
programs. Computer program methods to determine identity between two sequences include, but are
not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387
(1984)), BLASTP, BLASTN, and FASTA (Altschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990).
The BLAST X program is publicly available from NCBI and other sources (BLAST Manual,
Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:
403-410 (1990). The well known Smith Waterman algorithm may also be used to determine
identity.
Parameters for polypeptide sequence comparison include the following:
1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA.
89:10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4
A program useful with these parameters is publicly available as the "gap" program from Genetics
Computer Group, Madison WI. The aforementioned parameters are the default parameters for
peptide comparisons (along with no penalty for end gaps).
Parameters for polynucleotide comparison include the following:
1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3
Available as: The "gap" program from Genetics Computer Group, Madison WI. These are the
default parameters for nucleic acid comparisons.
A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be,
are provided in (1) and (2) below.
(1) Polynucleotide embodiments further include an isolated polynucleotide comprising a
polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the
reference sequence of SEQ ID NO:1,wherein said polynucleotide sequence may be identical to the
reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide
alterations as compared to the reference sequence, wherein said alterations are selected from the
group consisting of at least one nucleotide deletion, substitution, including transition and
transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of
the reference nucleotide sequence or anywhere between those terminal positions, interspersed either
individually among the nucleotides in the reference sequence or in one or more contiguous groups
within the reference sequence, and wherein said number of nucleotide alterations is determined by
multiplying the total number of nucleotides in SEQ ID NO: 1 by the integer defining the percent
identity divided by 100 and then subtracting that product from said total number of nucleotides in
SEQ ID NO:1, or:
nn ≤ xn - (xn • y),
wherein n n is the number of nucleotide alterations, x n is the total number of nucleotides in SEQ ID
NO:1, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%,
0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator,
and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to
subtracting it from x n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ
ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby
alter the polypeptide encoded by the polynucleotide following such alterations.
By way of example, a polynucleotide sequence of the present invention may be identical to
the reference sequence of SEQ ID NO: 1,that is it may be 100% identical, or it may include up to a
certain integer number of nucleic acid alterations as compared to the reference sequence such that
the percent identity is less than 100% identity. Such alterations are selected from the group
consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or
insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference
polynucleotide sequence or anywhere between those terminal positions, interspersed either
individually among the nucleic acids in the reference sequence or in one or more contiguous groups
within the reference sequence. The number of nucleic acid alterations for a given percent identity is
determined by multiplying the total number of nucleic acids in SEQ ID NO:1 by the integer defining
the percent identity divided by 100 and then subtracting that product from said total number of
nucleic acids in SEQ ID NO: 1, or:
nn ≤ xn - (xn • y),
wherein n n is the number of nucleic acid alterations, x n is the total number of nucleic acids in SEQ
ID NO: 1,y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., • is the symbol for the
multiplication operator, and wherein any non-integer product of x n and y is rounded down to the
nearest integer prior to subtracting it from x n. (2) Polypeptide embodiments further include an isolated polypeptide comprising a
polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide
reference sequence of SEQ ID NO:2, wherein said polypeptide sequence may be identical to the
reference sequence of SEQ ID NO: 2 or may include up to a certain integer number of amino acid
alterations as compared to the reference sequence, wherein said alterations are selected from the
group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative
substitution, or insertion, and wherein said alterations may occur at the amino- or
carboxy-terminal positions of the reference polypeptide sequence or anywhere between those
terminal positions, interspersed either individually among the amino acids in the reference sequence
or in one or more contiguous groups within the reference sequence, and wherein said number of
amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2
by the integer defining the percent identity divided by 100 and then subtracting that product from
said total number of amino acids in SEQ ID NO:2, or:
na ≤ xa - (xa • y),
wherein n a is the number of amino acid alterations, x a is the total number of amino acids in SEQ ID
NO:2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%,
0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator,
and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to
subtracting it from x a.
By way of example, a polypeptide sequence of the present invention may be identical to the
reference sequence of SEQ ID NO:2, that is it may be 100% identical, or it may include up to a
certain integer number of amino acid alterations as compared to the reference sequence such that the
percent identity is less than 100% identity. Such alterations are selected from the group consisting
of at least one amino acid deletion, substitution, including conservative and non-conservative
substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal
positions of the reference polypeptide sequence or anywhere between those terminal positions,
interspersed either individually among the amino acids in the reference sequence or in one or more
contiguous groups within the reference sequence. The number of amino acid alterations for a given
% identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the
integer defining the percent identity divided by 100 and then subtracting that product from said total
number of amino acids in SEQ ID NO:2, or:
na ≤ xa - (xa • y),
wherein n a is the number of amino acid alterations, x a is the total number of amino acids in SEQ ID
NO:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and • is the symbol for the
multiplication operator, and wherein any non-integer product of x a and y is rounded down to the
nearest integer prior to subtracting it from x a.
"Immunologically equivalent derivative(s)" as used herein encompasses a polypeptide,
polynucleotide, or the equivalent of either which when used in a suitable formulation to raise
antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction
between pathogen and mammalian host.
Immunospecific" means that characteristic of an antibody whereby it possesses substantially
greater affinity for the polypeptides of the invention or the polynucleotides of the invention than its
affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides
and polynucleotides in the prior art.
"Individual(s)" means a multicellular eukaryote, including, but not limited to a metazoan, a
mammal, an ovid, a bovid, a simian, a primate, and a human.
"Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature,
it has been changed or removed from its original environment, or both. For example, a polynucleotide
or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or
polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is
employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by
transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still
present in said organism, which organism may be living or non-living.
"Organism(s)" means a (i) prokaryote, including but not limited to, a member of the genus
Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria,
Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,
Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus.,
Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella,
Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella,
Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further
including, but not limited to, a member of the species or group, Group A Streptococcus, Group B
Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus
pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus. faecalis, Streptococcus
faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus,
Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium
tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes
israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella
bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus
aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi,
Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae,
Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,
Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus
cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidum,
Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon, including but not limited to
Archaebacter, and (iii) a unicellular or filamentous eukaryote, including but not limited to, a protozoan,
a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the
species Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans.
"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide,
which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include,
without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded
regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and
RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a
mixture of single- and double-stranded regions. In addition, "polynucleotide" as used herein refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions
may be from the same molecule or from different molecules. The regions may include all of one or
more of the molecules, but more typically involve only a region of some of the molecules. One of the
molecules of a triple-helical region often is an oligonucleotide. As used herein, the term
"polynucleotide(s)" also includes DNAs or RNAs as described above that contain one or more modified
bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are
"polynucleotide(s)" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual
bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are
polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications
have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or
metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells, including, for example, simple and complex cells.
"Polynucleotide(s)" also embraces short polynucleotides often referred to as oligonucleotide(s).
"Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to
each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains,
commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred
to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids.
"Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational
modifications, but also by chemical modification techniques. Such modifications are well
described in basic texts and in more detailed monographs, as well as in a voluminous research
literature, and they are well known to those of skill in the art. It will be appreciated that the same type
of modification may be present in the same or varying degree at several sites in a given polypeptide.
Also, a given polypeptide may contain many types of modifications. Modifications can occur
anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino
or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation,
amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of
a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation,
formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid
attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation,
selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such
as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND MOLECULAR
PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold,
F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic
Press, New York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) and Rattan et al., Protein
Synthesis. Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992).
Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched
circular polypeptides may result from post-translational natural processes and may be made by entirely
synthetic methods, as well.
"Recombinant expression system(s)" refers to expression systems or portions thereof or
polynucleotides of the invention introduced or transformed into a host cell or host cell lysate for the
production of the polynucleotides and polypeptides of the invention.
"Subtraction set" is one or more, but preferably less than 100, polynucleotides comprising
at least one polynucleotide of the invention
"Variant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a
reference polynucleotide or polypeptide respectively, but retains essential properties. A typical
variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide.
Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a
polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid
substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the
reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid
sequence from another, reference polypeptide. Generally, differences are limited so that the
sequences of the reference polypeptide and the variant are closely similar overall and, in many
regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or
more substitutions, additions, deletions in any combination. A substituted or inserted amino acid
residue may or may not be one encoded by the genetic code. The present invention also includes
include variants of each of the polypeptides of the invention, that is polypeptides that vary from the
referents by conservative amino acid substitutions, whereby a residue is substituted by another with like
characteristics. Typical such substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among
the acidic residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and Arg; or
aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2
or 1 amino acids are substituted, deleted, or added in any combination. A variant of a polynucleotide
or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is
not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides
may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods
known to skilled artisans.
EXAMPLES
The examples below are carried out using standard techniques, which are well known and
routine to those of skill in the art, except where otherwise described in detail. The examples are
illustrative, but do not limit the invention.
Example 1 Strain selection, Library Production and Sequencing
The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1 or 3] was
obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E. coli. The
sequencing data from two or more clones containing overlapping Staphylococcus aureus DNAs was
used to construct the contiguous DNA sequence in SEQ ID NO:1. Libraries may be prepared by
routine methods, for example:
Methods 1 and 2 below.
Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard
procedures and size-fractionated by either of two methods.
Method 1
Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate
according to standard procedures. DNA fragments of up to 11 kbp in size are rendered
blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are
ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard
procedures and E.coli infected with the packaged library. The library is amplified by standard
procedures.
Method 2
Total cellular DNA is partially hydrolyzed with a one or a combination of restriction
enzymes appropriate to generate a series of fragments for cloning into library vectors (e.g., RsaI,
Pall, AluI, Bsh1235I), and such fragments are size-fractionated according to standard procedures.
EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII
that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with
the packaged library. The library is amplified by standard procedures.
Example 2 gidA1 Characterization
The determination of expression during infection of a gene from Staphylococcus
aureus
Necrotic fatty tissue from a four day groin infection of Staphylococcus aureus WCUH29
in the mouse is efficiently disrupted and processed in the presence of chaotropic agents and
RNAase inhibitor to provide a mixture of animal and bacterial RNA. The optimal conditions for
disruption and processing to give stable preparations and high yields of bacterial RNA are
followed by the use of hybridisation to a radiolabelled oligonucleotide specific to Staphylococcus
aureus 16S RNA on Northern blots. The RNAase free, DNAase free, DNA and protein free
preparations of RNA obtained are suitable for Reverse Transcription PCR (RT-PCR) using unique
primer pairs designed from the sequence of each gene of Staphylococcus aureus WCUH29.
a) Isolation of tissue infected with Staphylococcus aureus WCUH29 from a mouse animal
model of infection
10 ml. volumes of sterile nutrient broth (No.2 Oxoid) are seeded with isolated, individual
colonies of Staphylococcus aureus WCUH29 from an agar culture plate. The cultures are
incubated aerobically (static culture) at 37°C for 16-20 hours . 4 week old mice (female, 18g-22g,
strain MF1) are each infected by subcutaneous injection of 0.5ml. of this broth culture of
Staphylococcus aureus WCUH29 (diluted in broth to approximately 108 cfu/ml.) into the anterior,
right lower quadrant (groin area). Mice should be monitored regularly during the first 24 hours
after infection, then daily until termination of study. Animals with signs of systemic infection, i.e.
lethargy, ruffled appearance, isolation from group, should be monitored closely and if signs
progress to moribundancy, the animal should be culled immediately.
Visible external signs of lesion development will be seen 24-48h after infection.
Examination of the abdomen of the animal will show the raised outline of the abscess beneath the
skin. The localised lesion should remain in the right lower quadrant, but may occasionally spread
to the left lower quadrant, and superiorly to the thorax. On occasions, the abscess may rupture
through the overlying skin layers. In such cases the affected animal should be culled immediately
and the tissues sampled if possible. Failure to cull the animal may result in the necrotic skin tissue
overlying the abscess being sloughed off, exposing the abdominal muscle wall.
Approximately 96 hours after infection, animals are killed using carbon dioxide
asphyxiation. To minimise delay between death and tissue processing /storage, mice should be
killed individually rather than in groups.The dead animal is placed onto its back and the fur
swabbed liberally with 70% alcohol. An initial incision using scissors is made through the skin of
the abdominal left lower quadrant, travelling superiorly up to, then across the thorax. The incision
is completed by cutting inferiorly to the abdominal lower right quadrant. Care should be taken not
to penetrate the abdominal wall. Holding the skin flap with forceps, the skin is gently pulled way
from the abdomen. The exposed abscess, which covers the peritoneal wall but generally does not
penetrate the muscle sheet completely, is excised, taking care not to puncture the viscera
The abscess/muscle sheet and other infected tissue may require cutting in sections, prior
to flash-freezing in liquid nitrogen, thereby allowing easier storage in plastic collecting vials.
b) Isolation of Staphylococcus aureus WCUH29 RNA from infected tissue samples
4-6 infected tissue samples(each approx 0.5-0.7g) in 2ml screw-cap tubes are removed
from -80°C.storage into a dry ice ethanol bath In a microbiological safety cabinet the samples are
disrupted individually whilst the remaining samples are kept cold in the dry ice ethanol bath. To
disrupt the bacteria within the tissue sample 1ml of TRIzol Reagent (Gibco BRL, Life
Technologies) is added followed by enough 0.1mm zirconia/silica beads to almost fill the tube, the
lid is replaced taking care not to get any beads into the screw thread so as to ensure a good seal and
eliminate aerosol generation. The sample is then homogenised in a Mini-BeadBeater Type BX-4
(Biospec Products). Necrotic fatty tissue isstrain treated for 100 seconds at 5000 rpm in order to
achieve bacterial lysis. In vivo grown bacteria require longer treatment than in vitro grown
Staphylococcus aureus Staphylococcus which are disrupted by a 30 second bead-beat.
After bead-beating the tubes are chilled on ice before opening in a fume-hood as heat
generated during disruption may degrade the TRIzol and release cyanide.
200 microlitres of chloroform is then added and the tubes shaken by hand for 15 seconds
to ensure complete mixing. After 2-3 minutes at room temperature the tubes are spun down at
12,000 x g, 4 °C for 15 minutes and RNA extraction is then continued according to the method
given by the manufacturers of TRIzol Reagent i.e.:- The aqueous phase, approx 0.6 ml, is
transferred to a sterile eppendorf tube and 0.5 ml of isopropanol is added. After 10 minutes at
room temperature the samples are spun at 12,000 x g, 4°C for 10 minutes. The supernatant is
removed and discarded then the RNA pellet is washed with 1 ml 75% ethanol. A brief vortex is
used to mix the sample before centrifuging at 7,500 x g, 4 °C for 5 minutes. The ethanol is
removed and the RNA pellet dried under vacuum for no more than 5 minutes. Samples are then
resuspended by repeated pipetting in 100 microlitres of DEPC treated water, followed by 5-10
minutes at 55°C. Finally, after at least 1 minute on ice, 200 units of Rnasin (Promega) is added.
RNA preparations are stored at -80°C for up to one month. For longer term storage the
RNA precipitate can be stored at the wash stage of the protocol in 75% ethanol for at least one
year at -20°C.
Quality of the RNA isolated is assessed by running samples on 1% agarose gels. 1 x TBE
gels stained with ethidium bromide are used to visualise total RNA yields. To demonstrate the
isolation of bacterial RNA from the infected tissue 1 x MOPS, 2.2M formaldehyde gels are run
and vacuum blotted to Hybond-N (Amersham). The blot is then hybridised with a 32P labelled
oligonucletide probe specific to 16s rRNA of Staphylococcus aureus ( K.Greisen, M. Loeffelholz,
A. Purohit and D. Leong. J.Clin. (1994) Microbiol. 32 335-351). The oligonucleotide of the
sequence is used as a probe. The size of the hybridising band is compared to that of control RNA
isolated from in vitro grown Staphylococcus aureus WCUH29 in the Northern blot. Correct sized
bacterial 16s rRNA bands can be detected in total RNA samples which show extensive degradation
of the mammalian RNA when visualised on TBE gels.
c) The removal of DNA from Staphylococcus aureus WCUH29-derived RNA
DNA was removed from 73 microlitre samples of RNA by a 15 minute treatment on ice
with 3 units of DNAaseI, amplification grade (Gibco BRL, Life Technologies) in the buffer
supplied with the addition of 200 units of Rnasin (Promega) in a final volume of 90 microlitres.
The DNAase was inactivated and removed by treatment with TRIzol LS Reagent (Gibco
BRL, Life Technologies) according to the manufacturers protocol.
DNAase treated RNA was resuspended in 73 microlitres of DEPC treated water with the addition
of Rnasin as described in Method 1.
d) The preparation of cDNA from RNA samples derived from infected tissue
10 microlitre samples of DNAase treated RNA are reverse transcribed using.a SuperScript
Preamplification System for First Strand cDNA Synthesis kit (Gibco BRL, Life Technologies)
according to the manufacturers instructions. 1 nanogram of random hexamers is used to prime each
reaction. Controls without the addition of SuperScriptll reverse transcriptase are also run. Both +/-RT
samples are treated with RNaseH before proceeding to the PCR reaction
e) The use of PCR to determine the presence of a bacterial cDNA species
PCR reactions are set up on ice in 0.2ml tubes by adding the following components: 45
microlitres PCR SUPERMIX (Gibco BRL, Life Technologies); 1 microlitre 50mM MgCl2, to
adjust final concentration to 2.5mM; 1 microlitre PCR primers(optimally 18-25 basepairs in length
and designed to possess similar annealing temperatures), each primer at 10mM initial
concentration; and 2 microlitres cDNA.
PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 as follows: 5
minutes at 95 °C, then 50 cycles of 30 seconds each at 94 °C, 42 °C and 72 °C followed by 3
minutes at 72 °C and then a hold temperature of 4 °C. (the number of cycles is optimally 30-50 to
determine the appearance or lack of a PCR product and optimally 8-30 cycles if an estimation of
the starting quantity of cDNA from the RT reaction is to be made); 10 microlitre aliquots are then
run out on 1% 1 x TBE gels stained with ethidium bromide with PCR product, if present, sizes
estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, Life Technologies). Alternatively
if the PCR products are conveniently labelled by the use of a labelled PCR primer (e.g. labelled at
the 5'end with a dye) a suitable aliquot of the PCR product is run out on a polyacrylamide
sequencing gel and its presence and quantity detected using a suitable gel scanning system (e.g.
ABI Prism™377 Sequencer using GeneScan™ software as supplied by Perkin Elmer).
RT/PCR controls may include +/- reverse transcriptase reactions, 16s rRNA primers or
DNA specific primer pairs designed to produce PCR products from non-transcribed
Staphylococcus aureus WCUH29 genomic sequences.
To test the efficiency of the primer pairs they are used in DNA PCR with Staphylococcus
aureus WCUH29 total DNA. PCR reactions are set up and run as described above using approx. 1
microgram of DNA in place of the cDNA and 35 cycles of PCR.
Primer pairs which fail to give the predicted sized product in either DNA PCR or RT/PCR
are PCR failures and as such are uninformative. Of those which give the correct size product with
DNA PCR two classes are distinguished in RT/PCR: 1.Genes which are not transcribed in vivo
reproducibly fail to give a product in RT/PCR; and 2.Genes which are transcribed in vivo
reproducibly give the correct size product in RT/PCR and show a stronger signal in the +RT
samples than the signal (if at all present) in -RT controls.
Two polynucleotide sequences of the invention, SEQ ID NOS: 1 and 3, were identified in
the above test as transcribed in vivo. SEQ ID NO:2 was deduced from the polynucleotide
sequence given as SEQ ID NO:1. SEQ ID NO:4 was deduced from the polynucleotide sequence
given as SEQ ID NO:3. The pair of PCR primers used to identify the gene are given as SEQ ID
NOS:5 and 6.
Annex to the description